1. Field of the Invention
The present invention relates in general to fluorescent pH sensors and ratiometric fluorescence measuring techniques. More particularly, the present invention relates to a fluorescent dual excitation-ratiometric pH sensor for non-invasive monitoring of pH.
2. Description of the Related Art
In recent years, there has been considerable research effort toward the development of techniques for continuous on-line monitoring of pH for environmental, biomedical and bioprocess applications. In particular, optical techniques based on fluorescence measurements offer many advantages over conventional electrochemical approaches including high sensitivity and ease of miniaturization. In addition, since fluorescence emission from an indwelling patch can be monitored without direct contact, in situ pH measurements can be made non-invasively with external instrumentation. The latter is highly desirable for bioprocesses using large, instrumented fermenters, by circumventing the cumbersome task of probe sterilization. For benchtop shake flasks and small scale, high throughput operations that do not readily accommodate the larger probes, in situ fluorescence-based patches delivering continuous real time pH values could be used to monitor and even control the acidity of the environment.
With judicious choice of the fluorescent indicator and immobilization conditions, measurements can be made over the desired region with high sensitivity. Sensors have been developed that measure the pH-dependent change in emission intensity, including those based on fluorescein, cyanine (1) and transition metal complexes (2-4). However, the inherent drawbacks of intensity-based measurements include signal variations due to probe photobleaching and fluctuations in source intensity, and sensitivity to position and orientation of the sensor patch that precludes non-invasive detection.
Furthermore, measurements in highly scattering or fluorescent media are difficult at best, even with an opaque membrane backing (5). Sensors based on the pH-dependent change in fluorescent lifetime of an immobilized ruthenium metal ligand complex (6) do not suffer the same drawbacks, but require more complicated instrumentation. In addition, sensitivity to collisional quenching by oxygen results in an additional calibration parameter or operation under anaerobic conditions.
An alternate approach that circumvents the problems associated with intensity-based measurements is ratiometric detection. Given a fluorescent indicator that exhibits a shift in excitation or emission wavelength with pH, the ratio of the emission intensity at the two wavelengths can be used as a robust measure of the pH that is insensitive to orientation, probe concentration and background fluorescence. Dual emission fiber-optic sensors based on seminapthofluorescein (7) and carboxynaphthofluorescein (8) have been described that rapidly and reliably correlate intensity ratios to pH. The extensive photobleaching that is observed for these dyes is accounted for by the ratiometric approach, but would still limit the useful lifetime of the sensor.
The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS) consists of a pyrene core with three sulfonic acid groups and a hydroxyl group that imparts pH sensitivity around a pka of approximately 7.3 (9). HPTS exhibits two excitation wavelengths, one UV at 405 nm and one blue at 457 nm, that correspond to the acid and its conjugate base (9-10). The subsequent pH-dependent shift in excitation maximum about the pKa of 7.3 enables dual-excitation ratiometric detection in the physiological range. This, together with a low toxicity (11) and insensitivity to oxygen concentration (12), makes HPTS a suitable probe for physiological and bioprocess pH measurements.
To date, covalent attachment of HPTS has been via sulfonamide coupling (13). The presence of the three strongly anionic sulphonic acid groups allows for HPTS to be immobilized by ionic binding to cationic supports. While effective in immobilizing the dye and preserving pH sensitivity, polymer substrates are limited to those that contain primary amines. In addition, amine groups which remain on the substrate after coupling will affect the local pH inside the polymer matrix. The dye has been covalently attached to controlled pore glass (15) and aminoethyl cellulose (16) in the development of fluorescence-based pH sensors that operate in neutral and acidic environments, as well as an intravascular blood gas monitoring system where it was used for both pH and pCO2 detection (17). Fiber-optic pH sensors have been described with HPTS bound to an anion exchange membrane (12) or resin (18) and fixed to the tip of the optical fiber.
For example U.S. Pat. No. 5,114,676 provides a pH sensor with a fluorescent indicator which may be covalently attached to a particle or to a microcrystalline cellulose fiber. The sensor comprises an optically transparent substrate, a thermoplastic layer and a hydrogel. Part of the particle with the indicator attached thereto is imbedded in a thermoplastic layer that is coated on the substrate and mechanically adhered using heat and pressure. The majority of the particle/indicator is imbedded within a hydrogel layer that is applied over the thermoplastic layer. The pH sensor is applied to the tip of an optical waveguide.
Furthermore, with the recent availability of low cost UV LEDS, the dye can be measured with relatively inexpensive instrumentation that combines UV and blue LEDs and a photodiode module. Such a setup has been described (14) to detect the pH of a high throughput microbioreactor system via HPTS directly dissolved in the fermentation media.
The inventors have recognized a need for improvement in HPTS sensors and uses thereof in inexpensively collecting ratiometric emission data externally from a biosystem. Specifically, the prior art is deficient in sensors comprising HPTS or derivatives thereof which is easily immobilized in a polymer matrix having suitable optical and diffusion properties. Additionally, the prior art is deficient in the use of substrates other than those having substituent primary amines. Furthermore, the prior art is deficient in sensors comprising HPTS or derivatives thereof which are easily assembled and which can be used as indwelling sensors in a reaction vessel to collect ratiometric emission data. The present invention fulfills this long-standing need and desire in the art.